![]() Normality was assessed with Shapiro-Wilk test. Statistical analysis was performed with Prism version 6 software (GraphPad, San Diego, CA, USA) and JMP Pro software version 11.2.0 (SAS, Carey, NC, USA). ![]() Prior to being mounted, cell nuclei were stained with Quinolinium, 4-1-, di-iodide (TO-PRO 3 Life Technologies, Grand Island, NY, USA). TUNEL staining was performed with ApopTag fluorescein direct in situ apoptosis detection kit (Millipore, Bedford, MA, USA), according to manufacturer's instructions. Eyes from these experimental models were enucleated, and 7- to 10-μm thin sections were cut using a model CM1850 cryostat (Leica Microsystems, Bannockburn, IL, USA). 13 To assess inner nuclear layer (INL) TUNEL + cells, we randomly selected 90 images from an experimental N-methyl- d-aspartic acid (NMDA)-induced excitotoxicity murine model that uses two different drug doses (10 and 100 nM). 12 For experimental comparison between groups, we selected 30 images from an experimental group (mammalian sterile 20-like kinase 2 knock-out mice) and compared them to a control group (C57BL/6 mice), as previously published. In order to quantitate TUNEL + cells on the outer nuclear layer (ONL), we randomly selected 90 images from an experimental murine retinal detachment model which induces photoreceptor cell death by subretinal injection of sodium hyaluronate. ![]() 3, 8 – 10 Despite its limitations, TUNEL assay remains the most widespread method used to screen for any form of programmed cell death, to date.ĭigital images of TUNEL-stained retinal cross-sections were obtained from the Angiogenesis Laboratory fluorescence microscopy database. 7 However, as TUNEL assay detects DNA fragments regardless of the induced cell death pathway, it cannot distinguish between apoptotic or other forms of programmed cell death. First described by Gavrieli et al., 6 this assay has been a sensitive method for detecting apoptosis. This assay detects fragmented or nicked DNA by means of a terminal deoxynucleotidyl transferase enzyme, which incorporates fluorescent-labeled dUTPs in damaged nucleic acid regions. 1 – 5 From the many tools used to characterize and quantify cell death, the terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay is likely the most widespread method. ![]() Several experimental animal models of diabetic retinopathy, retinitis pigmentosa, retinal detachment, and glaucoma, have revealed that cell death in the retina can occur through different modalities, including apoptosis and necroptosis among others, and encompasses multiple inducers and pathways and displays different morphologic characteristics. In recent years, research of degenerative retinal diseases has been focused on understanding cell death and ways of interfering with it. We believe that this standardized measurement tool could be advantageous to compare results across different research groups, as it is freely available as open source. We developed and validated an ImageJ macro that can be used as an accurate and precise quantitative tool for retina researchers to achieve repeatable, unbiased, fast, and accurate cell death quantitation. Comparing observers' results with macro results, COV was 23.37 ± 15.97% for the ONL and 23.44 ± 18.56% for the INL. The COV between both observers was 51.11 ± 25.83% for the ONL and 56.07 ± 24.03% for the INL. The intraobserver coefficient of variation (COV) ranged from 13.09% to 25.20%. Automated TUNEL + cell counts were in-between counts of inexperienced and experienced observers. The automated macro segmented outer nuclear layer (ONL) and inner nuclear layer (INL) successfully. To validate this tool, we selected a dataset of TUNEL assay digital images, calculated layer area and cell count manually (done by two observers), and compared measurements between observers and macro results. The script was coded using IJ1 programming language. We developed an ImageJ macro to quantitate cell death by TUNEL assay in retinal cross-section images. In this paper, we describe an automated quantification approach to address these difficulties. This process is time consuming, prone to measurement errors, and not entirely reproducible. Quantification of TUNEL-positive (TUNEL +) cells in tissue sections is usually performed manually, ideally by two masked observers. TUNEL assay is widely used to evaluate cell death.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |